BIOL 206: Introduction to Genetics Eric T. Rush, MD, FAAP, FACMG Clinical Geneticist Children’s Mercy Kansas City Associate Professor of Pediatrics University of Missouri – Kansas City School of Medicine Agenda

• Introduction to concepts and terminology • Description of how the information contained within DNA is used • Historical context and technology used to understand genetic changes What is genetics?

• Study of heredity and variation of essential character • Heredity – tendency of offspring to resemble their parents • Variation – tendency of offspring to vary from their parents • The term “genetics” was first coined in 1905 to describe the study of heredity • Greek “genno”, γεννώ; “to give birth” Genetic Terminology

• GENOTYPE: genetic make-up/composition • PHENOTYPE: aspects of a person you can see • MUTATION: alteration in the genetic code from what is expected • Does not necessarily communicate a change is pathogenic, so we generally use the term “variant” • Benign vs. pathogenic variant • BENIGN: not responsible for identified effects • PATHOGENIC: responsible for identified effects • VARIANT OF UNCERTAIN SIGNIFICANCE: unclear if responsible for identified effects Genetic versus Genomic

• Often used interchangeably in the lay press, but don’t make this mistake!

• GENETIC testing refers to specific testing of areas of known function

• GENOMIC testing refers to evaluation of large segments across the entirety of genetic material which may or may not have known function What are Chromosomes?

• Chromosomes are the structures that store genetic information in the form of DNA • Humans have 46 chromosomes • 22 pairs = autosomes • 1 pair = sex chromosomes (Most phenotypic males have one X and one Y chromosomes, while most phenotypic females have two X chromosomes) Normal Female Karyotype What are DNA and RNA?

http://youngbloodbiology.wikispaces.com/file/view/chromosome1.jpg/111553413/chromosome1.jpg Nucleotides Transcription

• Messenger RNA (mRNA) is transcribed from a DNA template by RNA polymerase • 3 phases: initiation, elongation, and termination Translation mRNA molecules leaves the nucleus and travels to the ribosome mRNA sequence attaches to site in the ribosome tRNA molecules, each with a complementary anti-codon sequence to mRNA also bears a specific amino acid

Amino acids are released and incorporated into the growing peptide chain

After peptide completed, process terminates and is released from ribosome

Inheritance Patterns

• Autosomal Recessive • The gene in question is located on an autosome (not X-chromosome) • Both copies of a gene (from mother and father) must have a variant (mutation) that reduces or eliminates normal function to have a disease • A person with such a change to one copy of a gene will typically be healthy, but can pass on to a child • Autosomal Dominant • The gene in question is located on an autosome • Only one copy of a gene must have a variant that alters normal function to have a disease Recurrence Risk and Epidemiology

• When both parents are carriers for a disease, the risk of a child inheriting the copy of the altered gene from both parents is 1 in 4 or 25% Recurrence Risk and Epidemiology

• When both parents are carriers for a disease, the risk of a child inheriting a copy of the altered gene from one, but not both parents is 1 in 2 or 50% Recurrence Risk and Epidemiology

Q: Why is it that most families with ZSD don’t have a known family history if the risk is 1 in 4? A: Because the 1 in 4 risk only comes into play when two carriers have already formed a mating pair. The odds are against that pair meeting in the first place. Time out for Terminology

• Homozygous – both copies of a gene have the same genetic change (e.g. PEX1 p.Gly843Asp/Gly843Asp) • Compound Heterozygous – both copies of a gene are altered, but the change itself is different (e.g. PEX1 p.Gly843Asp/p.Arg135Ter) • Heterozygous – one copy of a gene is altered (e.g. PEX1 p.Gly843Asp/WT) • “Carrier” is a term often used to describe heterozygous state in recessive disease DNA Sequencing

• The process of reading the order of the nucleotide bases in a DNA molecule

• Sequence alteration: Changes in the genetic code at the base pair level

Control: AAAGGCGCGTATAGGC Patient: AAAGGCGCGCATAGGC The Humble Beginning Human Genome Project

• “Eric, I need you to be as specific as possible about the ROI. We pay by the base pair!” • Email received by me Fall 1999

Approximate cost per megabase in 1999: $ 13,000

Chromosome 22, the smallest human chromosome, successfully sequenced Sanger Sequencing

• Also known as “chain terminator sequencing” • Oligonucleotide primers complementary to a template are used with each of the four nucleotides • DNA polymerase is used to extend the sequence • Chain terminating nucleotides are placed in low concentration as well • Usually dideoxynucleotides with a fluorescent dye tag Sanger Sequencing

• Differently sized fragments result and are terminated at the position that the dideoxynucleotide is incorporated • Fluorescent dye can be detected Sanger Sequencing

• Identification of changes at the base pair (DNA) level in one gene of interest 1. Useful when a specific genetic syndrome or disorder is suspected based on clinical findings and/or family history 2. Most diagnostically useful and cost-effective when a familial mutation is already known, permitting targeted mutation analysis Moore’s Law and Genetic Testing

In 1965, Gordon Moore observed that the number of components in an integrated circuit doubled every year, which was amended in 1975 to every two years, and held true until about 2012

Cost of sequencing mirrored Moore’s law until 2008, and since then has progressively exceeded it

Why? Next Generation Sequencing 1980 Automated 1990 sequencers have In the mid-1990’s, 2000 been available since new technology such the mid-1980’s using In 2004, 454 Life as pyrosequencing Sanger technology Sciences marketed a became available version of this for clinical use Exome Sequencing

• Sequencing of all coding regions of all genes • Estimated that 85% of all mutations will be in the exome • Cost of exome sequencing: • June 2010: $2,500 through a few centers • June 2012: $999 • January 2013: $698 • February 2016: $399 • What’s the catch? • Whole exome isn’t really whole exome • You get what you pay for Why do we use exome?

• Patient’s history and physical examination strongly suggests genetic etiology, but does not identify the specific etiology • The patient has had extensive evaluation, usually including several rounds of genetic testing without forthcoming diagnosis • Preferably, both parental specimens will be collected at the same time as the proband Limitations of Exome Sequencing

• Whole exome is really only about 85-90% of the total coding region • Mutations in non-coding region still can be important for disease • Human genome is massively variable and much we don’t understand • Of the 20,687 presently understood protein-coding genes, human disease is only presently described in 3,834 Whole Genome Sequencing (minus interpretation)

Cost of whole genome sequencing 1990-2003: $ 2,700,000,000 (Human Genome Project) 2004-2007: $ 100,000,000 (J. Craig Venter) January-April 2008: “less than $1.5 million” (James Watson) June 2009: $ 48,000 June 2010: $ 19,500 February 2013: $ 8,500 April 2016: $1,500 June 2020: $599